This proposal is directed at the Phase II development of qualitative and quantitative affinity-based mass spectrometry. Essentially, micro-scale immunoaffinity capture is used to selective isolate and concentrate given proteins from complex mixtures, after which matrix-assisted laser desorption/ionization time-of-flight mass spectrometry is used for sensitive and highly-accurate detection. The approach is further improved by the inclusion of internal standards, e.g., mass-shifted variants of the target proteins, resulting in a viable mass spectrometric immunoassay (MSIA) capable of the rigorous quantitation of proteins present in biological mixtures at sub-nanomolar concentrations. The specific aims of this proposal are to expand upon our Phase I findings by: 1) Further developing the devices needed for MSIA (MSIA-Tips) by increasing binding capacity and 2) improving surface derivatization methods, 3) Developing a "practice" MSIA kit for educational purposes, 4) Using MSIA with high-throughput robotics and mass spectrometers, and, 5) Increasing production of MSIA- Tips to meet projected demand at the end of Phase II. Mass spectrometric immunoassay (and associated devices and methods) is viewed as a relatively easy approach for the rapid qualitative and quantitative analysis of proteins, and in the study of protein-protein interactions, and thus stands to have significant impact in health-related research associated with protein characterization. PROPOSED COMMERCIAL APPLICATIONS: Potential applications of MSIA include routine screening of expression systems to verify production of recombinant proteins, protein/gene identification of genomic libraries via MS-database search techniques, and the rigorous quantitation of proteins present in natural biological mixtures. The commercial potential of the approach stems from lines of consumables (MSIA-Tips) that can be used singularly in research or in parallel for high- throughput applications and methods/techniques with applications in proteomics or the clinical/diagnostic arena.